To more completely assess the fungal species to which people are exposed, a fungal ribosomal RNA (rRNA) gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States indoor built environments are much more diverse than previously estimated using traditional methods of analysis. In FY16 fungal rRNA sequencing data from samples collected from a variety of geographic regions within the United States including Atlanta (Georgia), Bennington (Vermont), and Philadelphia (Pennsylvania) were analyzed using Illumina miSeq high throughput sequencing or Sanger sequencing to characterize fungal diversity in air and dust samples. In air and dust samples collected from the Atlanta and Bennington study sites the diversity of fungi is primarily composed of species placed in the phylum Ascomycota. Compared to viable culture datasets collected from the same location, species within the phylum Basidiomycota, Zygomycota, Glomeromycota, and Chytridiomycota were additionally resolved. The Basidiomycota species and causative agent of loose smut, Ustilago syntherismae, as well as Cryptococcus yeast species were the most abundant fungi identified in the Bennington analysis. The results of these ITS rRNA gene sequencing studies have provided new insights into the spectrum of fungi present in dust samples derived from occupational environments. As part of a broader Philadelphia school study to evaluate dampness and mold contamination in approximately 50 Philadelphia district schools NIOSH extracted and prepared 310 dust samples for Illumina MiSeq sequencing. These samples have been sequenced and the fungal diversity datasets will be analyzed in FY17. Additional studies are examining the molecular diversity of fungal contaminants as part of the NYC Neighborhood Asthma and Allergy Study. Preliminary analysis of analyzed dust samples indicates that Aureobasidium pullulans, Penicillium glabrum, Wallemia sebi and Alternaria alternata varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. Preliminary results suggest that multiple environmental factors including anthropogenic behavior modification, housing type, and neighborhood are important variables that influence fungal diversity within middle-income homes in New York City. A. alternata measured in house dust was also associated with fractional exhaled nitric oxide, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation. Fungal diversity captured using Illumina miSeq was compared to results obtained using qPCR. Work has continued in the development of antibodies to recombinant fungal biomarker antigens. The utility of these antibodies is critical for the quantification of fungal biomarkers, particularly to those fungi that are being studied by the NTP. NIOSH has recently developed a recombinant Alt a 1 homologue from Ulocladium chartarum tentatively named Ulo c 1. Polyclonal antibodies (pAb) have been produced toward the recombinant Ulo c 1. In FY16, pAb reactivity with rUlo c 1 as well as cross-reactivity with other Alt a 1 homologs has been determined and sandwich and inhibition ELISAs developed. Epitope mapping experiments are planned in FY17 to identify pAb affinity and specific epitopes.